2.5 In vitro wounding assay
A previously described wound healing scratch assay was used to assess
cell migration (Das et al. , 2018). Cells were plated at a density
of 160000 cells/ml in 24-well culture plates and incubated at 37 °C in
5% CO2 until formation of a confluent monolayer. Cells
were then scratched once per well, vertically, with a 200 μL plastic
pipette tip to create an artificial wound. After washing twice to remove
cellular debris by PBS, cells were treated with 10
ng·mL-1 FGF2, PEG-FGF2 conjugates, or PBS control. The
same fields were photographed every 12 h by Nikon Eclipse Ni light
microscopy and the areas of cell migration were calculated with ImageJ
Software (National Institutes of Health, Bethesda, MD, USA). The percent
migration at each time point was defined as the ratio of (the original
area - the open area at 24 h) to the original area. All the experiments
were performed in triplicate.